Gene therapy is used to insert genes into a patient's cells or tissues to treat hereditary diseases, whereby a defective mutant allele can be replaced by a functional one. Though the technology is still in its beginning and has been used with little success, it is promising for the future.
In 1990, the first approved gene therapy was performed at the U.S. National Institutes of Health on a four-year old girl. She was born with a rare genetic disease, called severe combined immunodeficiency (SCID). Children with this illness usually develop overwhelming infections and rarely survive to adulthood. In this first gene therapy, white blood cells were removed, cultivated and the missing gene was inserted into these cells. The genetically modified blood cells were reinfused into the patient's bloodstream (Anderson et al., 1990). Laboratory tests have shown that the therapy strengthened the immune system, but this procedure is not a cure. The genetically treated white blood cells only are functional for a few months, after which the procedure must be repeated.
The biology of human gene therapy is very complex, and there are many techniques that still need to be developed before gene therapy can be used appropriately. Scientists took the step of trying to introduce genes directly into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia (Nienhuis et al., 2003). However, this has been much harder than modifying simple bacteria, primarily because of the problems involved in carrying large sections of DNA and delivering them to the correct site on the comparatively large human genome. To deliver a therapeutic gene to a patient's target cells, a carrier of genetic material, e.g. a vector must be used. The most common types of vectors are viruses that have been genetically altered to loose e.g. their pathogenity. Retroviruses are suitable carriers, because they invert the genetic flow of information by reverse transcriptase-mediated conversion of their RNA genome into DNA and physically insert their genes into the host's genome by the enzyme integrase.
However, numerous problems exist that impede gene therapy using viral vectors, such as undesired side effects. For example, it has to be ensured that the virus will infect the correct cellular target and that the inserted gene does not disrupt any vital genes in the human genome. If the transduced gene is inserted into genes regulating cell division uncontrolled cell growth, i.e. cancer can occur by activation of oncogenes (Li et al., 2002; Check, 2005). Gene therapy trials to treat SCID were halted or restricted in the USA, when leukemia was reported in three of eleven patients treated in the French Therapy X-linked SCID gene therapy trial.
Viruses have natural host cell populations that they infect most efficiently, wherein retroviruses have limited natural host cell ranges. Attachment to and entry into a susceptible cell is mediated by the envelope polypeptide on the surface of a virus. Therefore, entry into potential host cells requires a favorable interaction between a protein on the surface of the virus and a protein on the surface of the host cell. For the purposes of gene therapy, one might either want to shift, limit or expand the range of cells susceptible to transduction by a gene therapy vector. To this end, many vectors have been developed in which the endogenous viral envelope proteins have been replaced by either envelope proteins from other viruses or by chimeric proteins. Viruses in which the envelope proteins have been replaced are referred to as pseudotyped viruses. For example, a popular retroviral vector for use in gene therapy trials has been the lentiviral simian immunodeficiency virus (Li et al., 1998) as well as the human immunodeficiency virus both coated with an envelope protein from a vesicular stomatitis virus.
The foamy virus subgroup of retroviruses has attracted scientific interest, because of their unique replication strategy and their potential use as gene transfer vectors (Weiss, 1996). It has been proposed that foamy viruses may be ideal tools for the development of a gene delivery system, due to specific properties of this virus group, such as the benign course of natural foamy viral infections, their very broad host cell range, and an extended packaging limit, due to the size of the foamy virus genome (Russel and Miller, 1996; Schmidt and Rethwilm, 1995; U.S. Pat. No. 5,929,222; U.S. Pat. No. 6,111,087). However, limited availability of improved foamy virus envelope genes has so far not allowed developing methods for preparing pseudotyped viral vectors that efficiently transfer genes into a wide variety of cell types.
Therefore, there is a demand for new nucleic acids, polypeptides and methods that improve efficiency of preparing pseudotyped vector particles and improve efficiency of transduction.
The solution to this problem is achieved by the embodiments of the present invention characterized by the claims, and described further below.